New anticancer agents are still in great demand due to the heterogeneous nature of cancer and the development of resistance to existing drugs. This collaborative proposal by three PIs (Richard Cerione, Hening Lin, and Robert Weiss) aims to establish SIRT5 inhibition as new strategy to treat cancers. SIRT5 is a member of the sirtuin family of enzymes that are known to have NAD+-dependent protein lysine deacylation activities. They play important roles in physiology, including the regulation of transcription, metabolism, and lifespan. We were the first to discover that SIRT5, a mitochondrial sirtuin with very weak deacetylation activity, prefers to hydrolyze negatively charged acyl lysine modifications, such as succinyl and malonyl lysine, from proteins. We and others have established lysine succinylation as an abundant post-translational modification that affects many metabolic enzymes. Moreover, we have recently obtained exciting preliminary data showing that SIRT5 deletion impairs tumorigenesis and metastasis in mice. At the cellular level, we found that inactivating SIRT5 inhibits the anchorage-independent growth of cancer cells, but in some cases has little effect on proliferation in conventional monolayer cultures. We hypothesize that SIRT5-mediated regulation of metabolism is required for malignant transformation and the acquisition of properties like anchorage- independent growth and invasiveness. One objective of the proposed studies is to understand the detailed molecular functions of SIRT5 in cancer cells, as well as in the tumor microenvironment. Successful completion of this goal is likely to shed light on the unique dependencies of cancers on metabolic alterations, knowledge that can help the development of novel therapeutics for treating cancer. Based on the discovery of the novel enzymatic activity of SIRT5, we have developed small molecule inhibitors of SIRT5 that are very selective and do not inhibit other sirtuins. Some of the inhibitors have shown promising anticancer activity in cell culture and mouse models. Another goal of this proposal is to develop additional SIRT5 inhibitors with improved in vivo efficacies against cancer in mouse models. We are very excited about the robust translational potential of the project, which is based on strong fundamental understanding of the enzymatic activity and biochemistry of SIRT5, coupled with rigorous biological analyses in cultured cells and mice.